Croy Carrie Hughes, Bergqvist Simon, Huxford Tom, Ghosh Gourisankar, Komives Elizabeth A
Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0378, USA.
Protein Sci. 2004 Jul;13(7):1767-77. doi: 10.1110/ps.04731004.
The crystal structure of IkappaBalpha in complex with the transcription factor, nuclear factor kappa-B (NF-kappaB) shows six ankyrin repeats, which are all ordered. Electron density was not observed for most of the residues within the PEST sequence, although it is required for high-affinity binding. To characterize the folded state of IkappaBalpha (67-317) when it is not in complex with NF-kappaB, we have carried out circular dichroism (CD) spectroscopy, 8-anilino-1-napthalenesulphonic acid (ANS) binding, differential scanning calorimetry, and amide hydrogen/deuterium exchange experiments. The CD spectrum shows the presence of helical structure, consistent with other ankyrin repeat proteins. The large amount of ANS-binding and amide exchange suggest that the protein may have molten globule character. The amide exchange experiments show that the third ankyrin repeat is the most compact, the second and fourth repeats are somewhat less compact, and the first and sixth repeats are solvent exposed. The PEST extension is also highly solvent accessible. Ikappa Balpha unfolds with a T(m) of 42 degrees C, and forms a soluble aggregate that sequesters helical and variable loop parts of the first, fourth, and sixth repeats and the PEST extension. The second and third repeats, which conform most closely to a consensus for stable ankyrin repeats, appear to remain outside of the aggregate. The ramifications of these observations for the biological function of IkappaBalpha are discussed.
与转录因子核因子κB（NF-κB）结合的IκBα的晶体结构显示出六个均有序排列的锚蛋白重复序列。尽管PEST序列中的大多数残基对于高亲和力结合是必需的，但未观察到其电子密度。为了表征未与NF-κB结合时IκBα（67-317）的折叠状态，我们进行了圆二色性（CD）光谱、8-苯胺基-1-萘磺酸（ANS）结合、差示扫描量热法和酰胺氢/氘交换实验。CD光谱显示存在螺旋结构，这与其他锚蛋白重复序列蛋白一致。大量的ANS结合和酰胺交换表明该蛋白可能具有熔球态特征。酰胺交换实验表明，第三个锚蛋白重复序列最紧密，第二个和第四个重复序列稍欠紧密，第一个和第六个重复序列暴露于溶剂中。PEST延伸部分也极易被溶剂接触。IκBα在42℃时展开，并形成一种可溶性聚集体，该聚集体隔离了第一个、第四个和第六个重复序列的螺旋和可变环部分以及PEST延伸部分。第二个和第三个重复序列与稳定的锚蛋白重复序列的共识最为接近，似乎保留在聚集体之外。讨论了这些观察结果对IκBα生物学功能的影响。
